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Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human β-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human β-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.

Hypertrophic cardiomyopathy–causing mutations disrupt a key regulatory off state of myosin in thick filaments., Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin and myosin binding protein-C (MyBP-C) lead to hypercontractility of the heart, an early hallmark of HCM. We show that hypercontractility caused by the HCM-causing mutation R663H cannot be explained by changes in fundamental myosin contractile parameters, much like the HCM-causing mutation R403Q. Using enzymatic assays with purified human β-cardiac myosin, we provide evidence that both mutations cause hypercontractility by increasing the number of functionally accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of MyBP-C. Furthermore, addition of C0-C7 decreases the wild-type myosin basal ATPase single turnover rate, while the mutants do not show a similar reduction. These data suggest that a primary mechanism of action for these mutations is to increase the number of myosin heads functionally available for interaction with actin, which could contribute to hypercontractility.

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif (IHM) lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, which could lead to hypercontractility at the molecular level., Hypertrophic cardiomyopathy (HCM) leads to hyper-contractility of the heart and is often caused by mutations in human β-cardiac myosin. Here authors show that four separate β-cardiac myosin mutations can modulate myosin activity by disrupting intramolecular interactions.

Hypertrophic cardiomyopathy (HCM) mutations in ß-cardiac myosin and myosin binding protein-C (MyBP-C) cause hypercontractility of the heart. We show that hypercontractility caused by the HCM myosin mutation R663H cannot be explained by changes in the fundamental parameters such as actin-activated ATPase, intrinsic force, velocity of pure actin or regulated thin filaments, or the pCa50 of the velocity of regulated thin filaments. The same conclusion was made earlier for the HCM myosin mutation R403Q (Nag et al. 2015). Using enzymatic assays for the number of functionally-available heads in purified human ß-cardiac myosin preparations, we provide evidence that both R403Q and R663H HCM myosin mutations cause hypercontractility by increasing the number of functionally-accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of myosin binding protein-C.

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, thereby leading to hypercontractility at the molecular level.

Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor-acceptor pairs to examine the kinetics of lever arm swing during the recovery and power stroke phases of the ATPase cycle. During the recovery stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the power stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-Å change in distance between the pre- and postpower stroke states and shows that the lever arm is more dynamic in the postpower stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid power stroke that gates the release of phosphate, whereas a second slower power stroke may be important for mediating strain sensitivity.

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

For more than 50years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

Summary:Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity and ATPase activityof myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative modelpositsthat mutations in myosin affect the stability ofa sequestered, super-relaxed state (SRX) of the proteinwith very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here, using a combination of biochemical and structural approaches, we show that purified myosin enters aSRX thatcorresponds to a folded-back conformation, which in muscle fibersresults insequestration of heads around the thick filament backbone. Mutations that cause HCM destabilize this state, while the small molecule mavacamtenpromotes it. These findings provide a biochemical and structural link between the genetics and physiology ofcardiomyopathywith implications for therapeutic strategies.

A working model for β-cardiac myosin in the sequestered state and binding assays reveal interactions between the myosin head and tail that are disrupted by mutations associated with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.

The sarcomere is an exquisitely designed apparatus that is capable of generating force, which in the case of the heart results in the pumping of blood throughout the body. At the molecular level, an ATP-dependent interaction of myosin with actin drives the contraction and force generation of the sarcomere. Over the past six decades, work on muscle has yielded tremendous insights into the workings of the sarcomeric system. We now stand on the cusp where the acquired knowledge of how the sarcomere contracts and how that contraction is regulated can be extended to an understanding of the molecular mechanisms of sarcomeric diseases, such as hypertrophic cardiomyopathy (HCM). In this review we present a picture that combines current knowledge of the myosin mesa, the sequestered state of myosin heads on the thick filament, known as the interacting-heads motif (IHM), their possible interaction with myosin binding protein C (MyBP-C) and how these interactions can be abrogated leading to hyper-contractility, a key clinical manifestation of HCM. We discuss the structural and functional basis of the IHM state of the myosin heads and identify HCM-causing mutations that can directly impact the equilibrium between the 'on state' of the myosin heads (the open state) and the IHM 'off state'. We also hypothesize a role of MyBP-C in helping to maintain myosin heads in the IHM state on the thick filament, allowing release in a graded manner upon adrenergic stimulation. By viewing clinical hyper-contractility as the result of the destabilization of the IHM state, our aim is to view an old disease in a new light.

Hypertrophic cardiomyopathy (HCM), the most commonly occurring inherited cardiovascular disease, is primarily caused by mutations in human β-cardiac myosin and myosin binding protein-C. It has been thought that such mutations in myosin increase the intrinsic force of the motor, its velocity of contraction, or its ATPase activity, giving rise to hyper-contractility. We hypothesize that while these parameters are mildly affected by most myosin HCM-causing mutations, a major effect of a majority of myosin HCM mutations is likely to involve an increase in the number of myosin heads that are functionally accessible (Na) for interaction with actin in the sarcomere. We consider a model involving three types of interactions involving the myosin mesa and the converter domain of the myosin motor that hold myosin heads in a sequestered state, likely to be released in a graded manner as the demands on the heart increase: 1) the two myosin heads binding to one another, 2) one head binding to its own coiled-coil tail, and 3) the other head binding to myosin binding protein-C. In addition there is clear evidence of interaction between the coiled-coil tail of myosin and myosin binding protein-C. Experimentally, here we focus on myosin head binding to its own coiled-coil tail and to myosin binding protein-C. We show that phosphorylation of the myosin regulatory light chain and myosin binding protein-C weaken these respective associations, consistent with known enhancements of sarcomere function by these phosphorylations. We show that these interactions are weakened as a result of myosin HCM mutations, in a manner consistent with our structural model. Our data suggests a potential unifying hypothesis for the molecular basis of hyper-contractility caused by human hypertrophic cardiomyopathy myosin mutations, whereby the mutations give rise to an increase in the number of myosin heads that are functionally accessible for interaction with actin in the sarcomere, causing the hyper-contractility observed clinically.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.

Myosins are proposed to utilize a conserved structural mechanism to generate force in which small conformational changes in the active site result in a large swing of the lever arm or light chain binding region. The converter domain is a flexible region that provides a link between the catalytic domain and lever arm and is proposed to play a critical role in the allosteric communication between these two domains. We introduced the R712G mutation in the converter domain and examined the impact of this mutation on the structural and functional properties of myosin V. The mutation resulted in a 16% reduction in the maximum actin-activated ATPase rate and no change in the actin concentration at which the ATPase activity is one-half maximal (KATPase). The sliding velocities examined in the in vitro motility assay were very similar between WT and R712G MV. We have developed a novel FRET system in myosin V (MV) that allows examination of the dynamics of lever arm motion. We labeled MV 11IQ containing an N-terminal (NT) tetracysteine motif with the bisarsenical dye FlAsH (MV.NT.FlAsH). The first IQ motif of MV.NT.FlAsH was exchanged with QSY-9 labeled CaM, a non-fluorescent acceptor. We followed the motion of the lever-arm during the ATP binding (recovery stroke) and actin-activated product release (power stroke) steps using stopped-flow FRET. The R712G mutation reduced the rate of the recovery stroke by 23% while having little impact on the fast power stroke that occurs prior to phosphate release. Thus, a mutation in the converter domain can specifically impact the recovery stroke without altering the power stroke demonstrating different allosteric mechanisms are responsible for these two key structural transitions.

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

Background: Magnesium may be an important physiological regulator of myosin motor activity. Results: Mg2+ inhibits the ADP release rate constant in the subset of myosins examined and reduces actin affinity in the post-hydrolysis state in myosin V. Conclusion: Mg2+ alters contractile velocity without altering overall tension-generating capacity. Significance: Mg2+-dependent regulation of motor activity is conserved in myosin motors. We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, -cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg2+-dependent manner (0.3-9.0 mm free Mg2+) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg2+ in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg2+ in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg2+ coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg2+ concentrations, demonstrating that the ADP release rate constant is slowed by Mg2+ in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg2+ reduces actin affinity in the MADPP(i) state, although it does not change the rate of phosphate release. Therefore, the Mg2+ inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg2+-dependent alterations in actin binding. Overall, our results suggest that Mg2+ reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.

We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

The N-terminus is a highly variable region among the myosin superfamily and has been associated with alterations in rotation of the lever arm and step size. We examined the structural dynamics of the N-terminal region of myosin V using steady-state and stopped-flow FRET. We introduced a tetra-cysteine site at the extreme N-terminus of a Myosin V 1IQ construct and labeled it with a bisarsenical fluorescin derivative (FlAsH) (MV NT-FLAsH). Energy transfer between FlAsH and mant or deac labeled nucleotides (dmantATP, dmantADP and deacATP) was monitored. Steady-state FRET experiments with the mant-FlAsH pair demonstrated a high FRET state in the presence of dmantATP and a low FRET state in the presence of dmantADP. Sequential-mix, in which MV NT-FLAsH is first mixed with dmantATP, aged to allow formation of the M.ADP.Pi state, and then mixed with saturating actin allowed us to explore the actin-acitvated product release steps. Biphasic transients with rates corresponding to the fast and slow rate of dmantADP release were observed. The sequential mix experiments with deacATP yielded three phases; a fast, actin dependent, phosphate release phase followed by the two phases of ADP release. Our results suggest the FRET signal monitors a structural change in the N-terminus associated with the sequential release of products. A high FRET state upon binding of ATP is associated with formation of the pre-powerstroke state of the lever arm and a low or no FRET state in the presence of ADP suggests formation of the post-powerstroke state. Our results allow us to hypothesize that movement of the N-terminal domain follows the movement of the converter/lever arm. Further experiments will explore the dynamics of the N-terminal domain and examine how its motion correlates with the conformation of the nucleotide binding pocket and lever arm.

We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain sensitive step in many muscle and non-muscle myosins. We also explore essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time resolved FRET analysis demonstrates a temperature dependent reversible conformational change in the nucleotide binding pocket. Our kinetic results demonstrate that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP while the actin binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating this is the rate limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates the transition from the open to closed nucleotide binding pocket conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the nucleotide binding pocket and actin binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open nucleotide binding pocket conformation may be altered by lever arm position.

We have used FRET to examine the kinetics and thermodynamics of the structural changes associated with ADP release in myosin V, which is thought to be a strain sensitive step in many muscle and non-muscle myosins. We also use essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine the intrinsic flexibility and correlated motions. Our kinetic and steady-state FRET results demonstrate that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP while the actin binding cleft remains closed. Thermodynamic analysis of ADP binding to actomyosin V suggests the collision complex formation is driven by a large enthalpy change and a small change in entropy. The transition from the open to closed pocket actomyosin.ADP state is associated with a large unfavorable decrease in entropy, which suggests the closed pocket conformation is more rigid than the open pocket conformation. Although no crystal structure is available of the closed pocket myosin V.ADP state, our FRET analysis reveals that this conformation may be similar to the myosin V.ATP state. FIRST/FRODA analysis is consistent with these conclusions as the myosin V.ADP structure is more flexible than the Apo structure, while the myosin V.ATP structure is more rigid than myosin V.ADP. Principal component analysis demonstrates that opening and closing of the nucleotide binding pocket correlates with the motions of loop 1 and the transducer region in all three crystal structures. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate correlates with the pocket opening step, suggesting this is the rate limiting step in the ATPase cycle. Our results provide insight into the structural mechanism of strain-dependent ADP release in myosins.

We examined the functional role of the upper 50 kDa hypertrophic cardiomyopathy (HCM) loop in myosin V. Hypertrophic cardiomyopathy is caused by missense mutations in highly conserved regions of myo2β and one deadly mutation occurs in the HCM loop (R403Q). Since the R403Q mutation has been shown to enhance or decrease the ATPase activity and in vitro motility of myosin II, it may be expected that the HCM loop plays a role in actin-activated product release. In our previous work we correlated the conformational change associated with ADP release and maximum ATPase rate in myosin V using FRET analysis. We engineered the R403Q mutation at an analogous site in myosin V 1IQ (R378Q) so that we would be able to investigate the impact of the mutation on actin-activated product release, maximum ATPase rate, in vitro motility, and FRET in myosin V. The R378Q mutation reduces the maximum ATPase rate two-fold while it slightly enhances sliding velocity compared to wild-type MV 1IQ. Our results suggest the duty ratio may be reduced as a result of the R378Q mutation. We will directly examine both ADP-release and phosphate-release to evaluate this possibility. To examine the impact of the point mutation on structural dynamics we will determine if conformational changes in the nucleotide-binding pocket and actin-binding cleft are disrupted using our established FRET probes. Our studies further establish a strategy for examining the mechanism of product release in myosin using the three assays: FRET, ATPase, and motility.

Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides and IAEDANS actin. These FRET studies suggest that myosin V can adopt a conformation in which the nucleotide binding pocket and the actin binding cleft are in a closed conformation. Our studies suggest the upper 50 kDa domain may be highly flexible in certain nucleotide-states which allows tight binding to nucleotide and actin. Molecular geometric simulations demonstrate the upper 50 kDa domain is most flexible in the myosin V.ADP state, consistent with this state having a high affinity for ADP and actin. Currently, we examined the temperature dependence of the FRET signal between mantADP and MV FlAsH. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-37°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. We also found that FlAsH labeled G440A MV, a non-hydrolyzable mutant, has a similar temperature-dependent transition in the presence of mantATP. In contrast, the transition does not occur in the presence of mantADP.BeFx or with the non-hydrolyzable E44A MV mutant in the presence of mantATP. Our results suggest coordination of the gamma-phosphate of ATP rigidifies the upper 50 kDa domain which results in a weak actin affinity state (open actin binding cleft and closed nucleotide binding pocket). However, upon phosphate release the upper 50kDa domain becomes more flexible which allows myosin to adopt a conformation in which it has a high affinity for both nucleotide and actin (closed nucleotide binding pocket and actin binding cleft).

We examined the impact of temperature and free magnesium concentration on monomeric FlAsH labeled myosin V (MV FlAsH), dimeric myosin V (MV HMM), and dimeric fast skeletal muscle myosin II (SK HMM) using ATPase and motility assays. Our results indicate that MV HMM and SK HMM both have a linear dependence on temperature that is similar in both ATPase and motility assays. However, MV FlAsH contains a different temperature dependence in ATPase and motility assays suggesting its short lever arm may impart a high strain dependence in the motility assay. MV HMM and MV FlAsH are inhibited by high concentrations of magnesium in both ATPase and motility assays. The rate-limiting step in myosin V is known to be ADP release, which we demonstrate correlates well with the magnesium and temperature dependence of ATPase and motility assays. Interestingly, SK HMM exhibits magnesium inhibition in ATPase assays, but only a slight decrease is observed in the motility assay. In SK HMM the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while in motility assays it is controversial. Our results indicate that SK HMM is better described by an attachment limited model in the motility assay. Magnesium may reduce the duty ratio of SK HMM which alters ATPase activity but not velocity in the motility assay. Future experiments will determine if magnesium alters the actin binding and/or the product release steps in myosin V and skeletal muscle myosin. Myosin V contains a tyrosine (residue 439) in the switch II region, which is an alanine at the corresponding position in myosin II, suggesting this residue may play a key role in differentially altering magnesium coordination in the active site of myosins.

The impact of two switch II mutations, G440A and E442A, on the conformation of the nucleotide binding pocket and actin binding cleft were examined with temperature dependent FRET analysis of FlAsH labeled myosin V (MV FlAsH). E442A MV FlAsH, which abrogates the salt bridge between switch I and switch II, remains in a closed nucleotide binding pocket state at all temperatures between 4 and 35°C in the presence of ATP indicating a highly stable closed pocket similar to wild-type MV FlAsH. The G440A MV mutant prevents the formation of a highly conserved hydrogen bond to the gamma-phosphate of ATP, and is able to form a closed pocket conformation at 25°C similar to E442A and WT MV. In the presence of ATP,G440A MV FlAsH populates a closed cleft conformation while E442A MV FlAsH forms an open cleft conformation. Our results suggest the switch II region is not critical for formation of the closed nucleotide binding pocket conformation while it is critical for communicating the conformational changes from the nucleotide binding region to the actin binding cleft. These results are supported by essential dynamic analyses using FIRST/FRODA applied to the myosin V crystal structures. To compare our FRET analysis to the thermal unfolding profile of myosin V we examined alpha-helical content by circular dichroism (CD) spectrometry as a function of temperature. We observed a broad transition at lower temperatures and a steep transition at higher temperatures in WT MV FlAsH. Comparing our FRET results with CD will allow us to determine if the conformational changes are associated with changes in secondary structure. Our results are interpreted in the context of identifying communication pathways essential to the energy transduction pathway of myosin motors.

Previously we have demonstrated in fluorescence resonance energy transfer (FRET) studies that mant labeled nucleotides and IAEDANS actin can act as good donor probes for a FlAsH labeled acceptor site in the upper 50 kDa domain of myosin V. We examined the temperature dependence of the FRET signal between mantADP and MV WT FlAsH in the presence and absence of actin. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-35°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. However, the mutant E442A, which is incapable of hydrolyzing ATP, remains in a high FRET state (closed pocket) with mantATP bound in the presence or absence of actin. Our results suggest a more flexible conformation of myosin in the presence of ADP compared to ATP which allows myosin to populate two actomyosin.ADP state conformations. These results are supported by the lifetime FRET analysis, and by computational FIRST/FRODA analysis of the intrinsic flexibility found in different x-ray crystal structures. We also plan to explore the temperature dependent conformational dynamics of the actin binding cleft using the IAEDANS actin (donor) and MV FlAsH (acceptor) pair in the presence of ATP, ADP, and absence of nucleotide using steady state and lifetime based FRET measurements. Our results will provide critical insights into the mechanocoupling that may occur between the nucleotide-binding pocket and actin binding cleft in myosin motors.

We investigated how magnesium impacts key conformational changes in the nucleotide binding pocket of myosin V and how these alterations impact the mechanochemical cycle. The conformation of the nucleotide binding pocket was examined using our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. Our previous work demonstrated the rate limiting conformation change in the actomyosin V ATPase cycle is opening of the nucleotide binding pocket which precedes ADP release from the open state. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free magnesium concentrations (0-10 mM) and find that the highest activity occurs at 0.5 mM magnesium, while there is a 50-60% reduction in activity above 4 mM magnesium. We also demonstrate that the motor activity assayed by in vitro motility is similarly dependent on magnesium concentrations. Transient kinetic studies of mantADP binding/release with actomyosin V FlAsH demonstrate the equilibrium between the open and closed nucleotide binding pocket conformations is dependent on magnesium with the closed state stabilized by magnesium. We find that the kinetics of the nucleotide binding pocket opening step correlates well with the ATPase and motility results over a wide range of magnesium concentrations. In the absence of magnesium (presence of 4 mM EDTA) the nucleotide binding pocket populates a single conformation that is dramatically open at higher temperatures. In addition, magnesium significantly slows the rate of ADP release from the open state. Our results shed light on the structural mechanism of ADP release in myosin V and allow us to speculate about the conserved conformational pathways involved in strain sensitive ADP release in myosins.

Kinetic and structural studies of both muscle and non-muscle myosins have revealed that the enzymatic cycle of these motors frequently contains more than one actomyosin ADP state. Interestingly, the rate of ADP release in myosin motors is thought to be the main determinant of sliding velocity in muscle, suggesting strain dependent ADP release may be a critical mechanism of mechanochemical coupling. Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH (MV FlAsH) can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides. We also determined that this donor-acceptor pair likely monitors opening/closing of the nucleotide binding pocket. Currently, we utilized the FRET signal to examine the kinetics of nucleotide binding pocket opening during the process of mantADP release from acto-MV FlAsH. We obtained evidence that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP. We also explored the temperature dependence of the closed to open transition and nucleotide release steps. We find that at lower temperatures the closed conformation is favored while at higher temperature the open conformation is favored. The more rapid ADP release step which follows nucleotide binding pocket opening is also temperature dependent. Therefore, since both steps are temperature-dependent they likely require significant conformational changes. We also compared our FRET results to the rate of ATP-induced dissociation from actin in the presence of ADP monitored by light scatter. Understanding how strain alters either of these two steps may be critical for elucidating the structural mechanism of strain-dependent ADP release in myosins.